Geranyl diphosphate synthase genes

ABSTRACT

An isolated recombinant protein comprising the amino acid sequence shown in SEQ ID No: 1 is disclosed. A preferred embodiment of the invention is a recombinant protein having the amino acid sequence shown in SEQ ID NO: 1 but having a deletion, substitution or addition of at least one amino acid, excluding the amino acid at position 82, and which has geranyl diphosphate synthase activity. Also disclosed is the gene encoding the recombinant protein.

TECHNICAL FIELD

The present invention relates to a geranyl diphosphate synthase, a geneencoding the synthase, a recombinant vector comprising the gene, andmethods for preparing a geranyl diphosphate synthase and geranyldiphosphate, respectively.

BACKGROUND ART

Among those substances which have an important function in organisms,there are a large number of substances biosynthesized with isoprene(2-methyl-1,3-butadiene) units. These compounds are also calledisoprenoids, terpenoids or terpenes. Depending on the number of carbonatoms they have, they are classified into hemiterpene (C5), monoterpene(C10), sesquiterpene (C15), diterpene (C20), sesterterpene (C25),triterpene (C30), tetraterpene (C40) and the like.

Actual biosynthesis of these substances starts with the synthesis ofisopentenyl diphosphate (IPP), the active isoprene unit. Ultimately, theactual form of the isoprene unit which had been proposed as an putativeprecursor substance is IPP, the so-called active isoprene unit.

It is known that dimethylallyl diphosphate (DMAPP), an isomer of IPP, issynthesized into an active isoprenoid such as geranyl diphosphate (GPP),neryl diphosphate, farnesyl diphosphate (FPP), geranylgeranyldiphosphate (GGPP), geranylfarnesyl diphosphate (GFPP), hexaprenyldiphosphate (HexPP) or heptaprenyl diphosphate (HepPP), throughcondensation with IPP.

Through cis-condensation of FPP, GPP and the like in which the all-Etype is considered to be the active type, a number of compounds such asnatural rubber, dolichol, bactoprenol (undecaprenol) or variouspolyprenols found in plants are synthesized. It is considered that thesecompounds are synthesized by the consecutive condensation using theenergy of phosphate bonds between the pyrophosphoric acid and the carbonskeleton in their molecules. It is considered that pyrophosphoric acidis generated as a by-product of the condensation.

Active type isoprenoid synthases which condensate IPP into allylicsubstrates of DMAPP, GPP, FPP, GGPP, GFPP, etc. in succession are calledprenyl diphosphate synthases or prenyltransferases. Prenyl diphosphatesynthases have different designations depending on the number of carbonatoms in their major reaction product. For example, the enzyme whichcatalyzes the production of farnesyl diphosphate with 15 carbon atoms iscalled farnesyl diphosphate synthase (FPP synthase); and the enzymewhich catalyzes the production of geranylgeranyl diphosphate with 20carbon atoms is called geranylgeranyl diphosphate synthase (GGPPsynthase).

Various prenyl diphosphate synthase genes have already been obtainedfrom bacteria, archaea, fungi, plants and animals. Purification,activity determination as well as gene cloning and DNA sequencing havebeen reported on FPP synthases, GGPP synthases, hexaprenyl diphosphatesynthases, heptaprenyl diphosphate synthases, octaprenyl diphosphatesynthases, nonaprenyl diphosphate synthases (solanesyl diphosphatesynthases), undecaprenyl diphosphate synthases and the like.

These prenyl diphosphate synthases that are fundamental for thesynthesis of important and diversified compounds from both industrialand life-scientific viewpoints are generally unstable and low inspecific activity. Thus, industrial application of them could not beexpected. In recent several years, however, thermostable FPP synthasegenes and GGPP synthase genes have been isolated from thermophilicbacteria and archaea [A. Chen and D. Poulter (1993), J. Biol. Chem., 268(15), 11002-11007; T. Koyama et al., (1993), J. Biochem. (Tokyo), 113(3), 355-363; S.-i. Ohnuma et al., (1994), J. Biol. Chem., 269 (20),14792-14797]. Thus, conditions for utilizing prenyl diphosphatesynthases are now being prepared.

Enzymes which synthesize C₁₀₋₂₅ prenyl diphosphates are homodimers. Itis relatively easy to allow them to react in vitro, and a number ofreports have been made on their reaction. In those enzymes, an enzymehaving activity to synthesize GPP (a C₁₀ prenyl diphosphate)specifically has not been isolated, though partial purification of ithas been reported (L. Heide and U. Berger, 1989, Arch, Biochem.Biophys., 273 (2) 331-8). Although it has been reported that a GPPsynthase was successfully purified from pig liver (J. K. Dorsey et al.,1966, J. Biol. Chem. 241 (22), 5353-5360), this enzyme catalyzes thesynthesis of FPP at the same time. Thus, based on the current definitionof prenyl diphosphate synthase, this enzyme should be called FPPsynthase.

GPP is the first intermediate for the synthesis of many monoterpenesknown and is the most important compound in the biosynthesis pathway ofmonoterpenes.

Both geraniol and its isomer nerol, which are representativemonoterpenes, are aromatics in the major components of rose oil. Anotherrepresentative monoterpene camphor which is an extract from Cinnamomumcamphora is also used as a mothball.

However, GPP synthase gene has not been isolated yet.

Under circumstances, a technology is demanded which artificiallymodifies the amino acid sequence of a thermophile-derived, stable,homodimer type prenyl diphosphate synthase having a high specificactivity to thereby engineer a homodimer type, thermostable prenyldiphosphate synthase which specifically catalyzes the synthesis of GPP.

As thermophile-derived prenyl diphosphate synthases, Bacillusstearothermophilus FPP synthase and Sulfolobus acidocaldarius GGPPsynthase have been modified. Mutants of the S. acidocaldarius GGPPsynthase and genes thereof were selected using as an indicator anability to complement the glycerol metabolism ability of a HexPPsynthesis-dificient Saccharomyces serevisiae (budding yeast) [S.-i.Ohnuma et al., (1996), J. Biol. Chem., 271 (31), 18831-18837]. Mutantsof the S. stearothermophilus FPP synthase having GGPP synthesis activityand genes thereof were obtained using lycopene synthesis as an indicator[S.-i. Ohnuma et al., (1996), J. Biol. Chem., 271 (17), 10087-10095].Further, 18 mutant enzymes which synthesize a number of prenyldiphosphates from GGPP to HexPP in various proportions, and genesencoding those enzymes were obtained by site-directed mutagenesis thenucleotides encoding the amino acid residue located 5 amino acidresidues upstream of the Asp-rich domain conserved region I (DDXX(XX)D)[S.-i. Ohnuma et al., (1996), J. Biol. Chem., 271 (48), 30748-30754]. Ithas been found that the amino acid residue located 5 amino acid residuesupstream of the Asp-rich domain conserved region I (DDXX(XX)D) isinvolved in the regulation of chain lengths of reaction products.

However, no mutant enzyme having activity to synthesize GPP specificallyhas been obtained yet.

DISCLOSURE OF THE INVENTION

It is an object of the invention to provide a geranyl diphosphatesynthase and a gene encoding the synthase.

As a result of extensive and intensive researches toward the solution ofthe above problem, the present inventor has succeeded in isolating ageranyl diphosphate synthase and a gene encoding the synthase byreplacing a part of the amino acid sequence of a farnesyl diphosphatesynthase. Thus, the present invention has been achieved.

The present invention relates to the following recombinant protein (a)or (b):

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO:1;

(b) a protein which consists of the amino acid sequence shown in SEQ IDNO: 1 having deletion, substitution or addition of at least one aminoacid excluding the amino acid at position 82, and which has geranyldiphosphate synthase activity.

Further, the present invention relates to a gene coding for theabove-described recombinant protein (a) or (b).

Further, the present invention relates to a geranyl diphosphate synthasegene comprising the nucleotide sequence shown in SEQ ID NO: 2.

Further, the present invention relates to a recombinant vectorcomprising any of the above-described genes.

Further, the present invention relates to a transformant transformedwith the above-described recombinant vector.

Further, the present invention relates to a method of preparing ageranyl diphosphate synthase comprising culturing the above-describedtransformant in a medium and recovering the geranyl diphosphate synthasefrom the resultant culture.

Further, the present invention relates to a method of preparing geranyldiphosphate comprising culturing the above-described transformant in amedium and recovering geranyl diphosphate from the resultant culture.

Further, the present invention relates to a method of preparing geranyldiphosphate comprising allowing a culture of the above-describedtransformant to act on isopentenyl diphosphate or an isomer thereof.

Hereinbelow, the present invention will be described in more detail.

It is known that there are five conserved regions in the amino acidsequence of a prenyl diphosphate synthase (if the synthase is aheterodimer, in the amino acid sequence of one of the sub-unit) [A. Chenet al. (1994) Protein Sci., 3 (4), 600-607]. In these five conservedregions (conserved regions I-V), there are two regions rich in asparticacid residues to which reaction products or reaction substrates arebelieved to be bound. These regions are called “aspartic acid richdomains” or “Asp-rich domains”. Of these, the Asp-rich domain located atthe amino terminal of prenyl diphosphate synthases (i.e. located in theabove-mentioned conserved region II) is designated Asp-rich domain I[sequence: DDXX(XX)D wherein the XX in parentheses may not exist], andthe Asp-rich domain located at the carboxyl terminal (i.e. located inthe above-mentioned conserved region V) is designated Asp-rich domain IIfor the purpose of discrimination.

Specific examples of prenyl diphosphate synthases containing suchaspartic acid rich domains as described above include farnesyldiphosphate synthase, geranylgeranyl diphosphate synthase, hexaprenyldiphosphate synthase, heptaprenyl diphosphate synthase, octaprenyldiphosphate synthase, nonaprenyl diphosphate synthase and undecaprenyldiphosphate synthase. As more specific examples, Bacillusstearothermophilus farnesyl diphosphate synthase, Escherichia colifarnesyl diphosphate synthase, Saccharomyces cerevisiae farnesyldiphosphate synthase, rat farnesyl diphosphate synthase, human farnesyldiphosphate synthase, Saccharomyces cerevisiae hexaprenyl diphosphatesynthase and the like may be enumerated. The amino acid sequences of theabove-mentioned conserved regions I-V in bacterial farnesyl diphosphatesynthases among those examples are shown in FIG. 4. In FIG. 4, “1.”represents the amino acid sequence of Bacillus stearothermophilusfarnesyl diphosphate synthase and “2.” represents the amino acidsequence of E. coli farnesyl diphosphate synthase. The portion enclosedwith a larger box shows Asp-rich domain I, and the portion marked with“⋆” shows the amino acid residue located 4 amino acid residues upstreamof this Asp-rich domain I.

The present invention is characterized by the creation of a geranyldiphosphate synthase by substituting the amino acid residue located 4amino acid residues upstream of Asp-rich domain I with other amino acidresidue having a larger molecular weight than that residue, and by thepreparation of geranyl diphosphate through the enzyme reaction of theresultant geranyl diphosphate synthase. More specifically, a geranyldiphosphate synthase is created by substituting the amino acid residuemarked with “⋆” in FIG. 4 (Ser) located 4 amino acid residues upstreamfrom the N-terminal amino acid (Asp) of the sequence DDXX(XX)Dconstituting Asp-rich domain I with other amino acid residue having alarger molecular weight than Ser (any amino acid excluding Gly and Ala;i.e. any amino acid selected from the group consisting of Val, Leu, Ile,Thr, Asp, Glu, Asn, Gln, Lys, Arg, Cys, Met, Phe, Tyr, Trp, His andPro). The amino acid used for the above substitution is not particularlylimited as long as it is neither Gly nor Ala. Preferably, Phe is used.

Specifically, the geranyl diphosphate synthase of the invention is canbe obtained by substituting the Ser residue at position 82 of the aminoacid sequence shown in SEQ ID NO: 5 of a farnesyl diphosphate synthasewith, for example, Phe residue.

Such substitution can be achieved by partially modifying the nucleotidesequence of the gene encoding B. stearothermophilus FPP synthase whichis reported to be highly thermostable and high in specific activity.

(1) Preparation of a Target Gene for Mutagenesis

A target gene into which a mutation is to be introduced is the geneencoding Bacillus stearothermophilus FPP synthase (hereinafterabbreviated to “BstFPS”). The full length nucleotide sequence of theBstFPS gene is known [T. Koyama et al., (1993) J. Biochem., 113,355-363; SEQ ID NO: 4] and is disclosed under Accession No. D13293 ingenetic information databases such as DDBJ.

Since B. stearothermophilus is also available from various microorganismdepositories such as ATCC (ATCC 10149), the DNA of the BstFPS gene canbe obtained by conventional gene cloning methods [S. Sambrook et al.(eds.), (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press,New York].

Subsequently, the resultant DNA fragment is ligated to an appropriateplasmid vector (e.g. pTV118N from Takara Shuzo) to thereby prepare aplasmid DNA for mutagenesis. This plasmid DNA is designated pFPS.

(2) Synthesis of an Oligonucleotide for Mutagenesis and Introduction ofa Mutation

An oligonucleotide for mutagenesis is designed so that (a) the Ser codoncorresponding to the amino acid residue at position 82 of BstFPS issubstituted with any codon (such as Phe codon) corresponding to anyamino acid other than Gly, Ala and Ser; and (b) a restriction site forBspHI (5′TCATGA 3′) is newly introduced. For example, the followingnucleotide sequence may be given for the oligonucleotide.

5′-CAT ACG TAC TTC TTG ATT CAT GAT GAT TTG-3′ (SEQ ID NO: 6)

This nucleotide sequence is designed so that the amino acid sequenceencoded by the BstFPS gene is not altered by degeneracy of codons evenafter the introduction of the BspHI site. Because of the introduction ofthis restriction site, it is possible to detect those plasmids intowhich a substitution mutation has been introduced by agarose gelelectrophoresis of plasmid DNA after BspHI digestion.

The synthesis of the oligonucleotide may be performed with conventionalchemical synthesis equipment. Preferably, the synthesizedoligonucleotide is phosphorylated and then denatured (for example, byheating it at 70° C. for 10 min).

Subsequently, using the oligonucleotide as a primer, a mutation isintroduced into the plasmid prepared as described above. The method ofintroduction of a mutation is not particularly limited. For example., acommercial kit based on the method of Kunkel [Proc. Natl. Acad. sci.,USA (1985) 82, 488] (Mutan-K kit from Takara Shuzo) may be used.Alternatively, polymerase chain reaction (PCR) may be used.

A single-stranded DNA is prepared as a template, and then theoligonucleotide described above was annealed with the template as acomplementary strand synthesis primer DNA to thereby obtain adouble-stranded DNA. The resultant DNA is incorporated into a plasmid,with which an E. coli strain is transformed.

The gene of the invention can be easily obtained, for example, byintroducing a mutation into the DNA encoding the native amino acidsequence of the synthase (SEQ ID NO: 4) by a conventional method such assite-directed mutagenesis or PCR.

For the resultant transformant clones, their nucleotide sequences aredetermined. This determination may be performed by any conventionalmethod such as Maxam-Gilbert method or the dideoxy method. Usually, thedetermination is performed with an automated DNA sequencer based on thedideoxy method.

SEQ ID NO: 2 illustrates by way of example a nucleotide sequence for thegene of the invention. SEQ ID NOS: 1 and 3 illustrate by way of exampleamino acid sequences for the geranyl diphosphate synthase of theinvention, which sequences may have a mutation such as deletion,substitution or addition of at least one amino acid (e.g. one or severalamino acids) excluding the amino acid at position 82 (e.g. Phe) as longas the protein consisting of the mutated sequence has geranyldiphosphate synthase activity. For example, the amino acid sequence ofSEQ ID NO: 1 or 3 in which the Met at position 1 is deleted is alsoincluded in the geranyl diphosphate synthase of the invention. Also, thegenes encoding these geranyl diphosphate synthases are also included inthe gene of the invention.

The “geranyl diphosphate synthase activity” used herein means acatalytic activity to synthesize GPP using IPP or an isomer thereof(e.g. DMAPP) as a substrate. The introduction of a mutation may beperformed by the same method as described above.

Once the nucleotide sequence of the geranyl diphosphate synthase gene ofthe invention has been established, the gene of the invention can beobtained by chemical synthesis, or by PCR using the gene as a template,or by hybridization using a DNA fragment having a nucleotide sequence ofthe gene as a probe.

(3) Construction of a Vector

A recombinant vector of the invention can be obtained by ligating thegene of the invention into an appropriate vector. The vector into whichthe gene of the invention is to be inserted is not particularly limitedas long as it is replicable in a host. A vector which may be used forthe preparation of the recombinant vector of the invention can beprepared E. coli or the like by the alkali extraction method (Birnboim,H. C. & Doly, J. (1979) Nucleic Acid Res. 7: 1513) or a variationthereof. Alternatively, a commercial vector may be used as it is, orvarious vectors induced according to purposes may be used. For example,pBR322, pBR327, pKK233-2, pKK233-3 or pTrc99A having a pMB1-derivedreplication origin may be enumerated. In addition, pUC18, pUC19, pUC118,pUC119, pTV118N, pTV119N, pBluescript, pHSG298 or pHSG396 which ismodified to give a greater number of copies, or a plasmid derived frompSC101, ColE1 factor, R1 plasmid or F factor may be enumerated. Further,a fusion protein expression vector such as pGEX vector or pMal vectorwhich facilitates the purification of the expressed product may be used.

It is also possible to perform gene transfer using a virus vector (e.g.λ phage or M13 phage) or a transposon instead of a plasmid. As a phageDNA, M13mp18, M13mp19, λgt10, λgt11 or the like may be used.

The incorporation of a DNA fragment encoding the geranyl diphosphatesynthase into such a vector can be performed by conventional methodsusing an appropriate restriction enzyme and ligase. For example, amethod may be employed in which a purified DNA is digested with anappropriate restriction enzyme and then inserted into the relevantrestriction site of an appropriate vector DNA for ligation.

The gene of the invention should be incorporated in the vector in such amanner that the function of the gene can be manifested. For thispurpose, the vector of the invention may contain a replication originand expression regulating sequences appropriate for the host to be used.Further, the vector may also contain a transcription promoter,transcription terminator, ribosome binding site or the like. As thepromoter, Ptac, Plac or Ptrc may be used. As the terminator, rrnBterminator may be used. As the ribosome binding site, SD sequence(represented by 5′-AGGAGG-3′) may be used.

As a specific example of the thus prepared plasmid vector, pFPSmdescribed in Examples may be given.

(4) Preparation of a Transformant

A transformant of the invention can be obtained by introducing therecombinant expression vector of the invention into a host so that thegene of interest can be expressed.

The host to be used is not particularly limited as long as it canexpress the gene of the invention. Specific examples of the host includeEscherichia or Bacillus bacteria such as E. coli, B. subtilis, B.brevis; Saccharomyces or Pichia yeasts such as S. cerevisiae, P.Pastris; filamentous fungi of the genus Aspergillus such as A. oryzae,A. niger; cultured cells of silkworm; animal cells such as COS cells orCHO cells; or plant cells.

When a bacterium such as E. coli is used as the host, preferably, therecombinant vector of the invention is capable of autonomous replicationin the host and, at the same time, is composed of a transcriptionpromoter, a ribosome binding site, the DNA of the invention and atranscription terminator. The vector may also contain a gene to controlthe transcription promoter.

As the promoter sequence to start the transcription from DNA to mRNA, anative sequence (such as lac, trp, bla, lpp, PL, PR, T3 or T7) may beused. In addition to these promoters, mutants thereof (e.g. lacUV5) orsequences (e.g. tac, trc, etc.) in which a native promoter sequence isartificially fused are known and may be used in the present invention.

With respect to a sequence which regulates the ability to synthesize aprotein from mRNA, it is already known that the distance between theribosome binding site (GAGG and similar sequence) to the initiationcodon (ATG or GTG) is important. Further, it is well known that aterminator which commands the termination of transcription at the 3′ end(e.g. rrnBT1T2) influences upon the protein synthesis efficiency in arecombinant. Therefore, in the present invention, gene expression can beperformed efficiently by using these sequences.

As a method for introducing a foreign gene into a bacterium, any methodof DNA introduction into bacteria may be used. For example, a methodusing calcium ions [Proc. Natl. Acad. Sci., USA, 69:2110-2114 (1972)],electroporation or the like may be used.

When a yeast is used as the host, YEp13, YEp24, YCp50 or the like isused as an expression vector. As a promoter used in this case, anypromoter may be used as long as it can direct the expression of the geneof interest in yeasts. For example, gal1 promoter, gal10 promoter, heatshock protein promoter, MFα1 promoter or the like may be enumerated.

As a method for introducing a foreign gene into the yeast, any method ofDNA introduction into yeasts may be used. For example, electroporation[Methods Enzymol., 194:182-187 (1990)], the spheroplast method [Proc.Natl. Acad. Sci., USA, 84:1929-1933 (1978)], the lithium acetate method[J. Bacteriol., 153:163-168 (1983)] or the like may be enumerated.

When an animal cell is used as the host, pcDNAI/Amp, pcDNAI or the likeis used as an expression vector. In this case, the early gene promoterof human cytomegalovirus or the like may be used as a promoter.

As a method for introducing a foreign gene into the animal cell,electroporation, the calcium phosphate method, lipofection or the likemay be enumerated.

As a method for introducing a foreign gene into a plant cell, theinfection method using Agrobacterium is widely used. As a method fordirect introduction, the protoplast method, electroporation,bombardment, etc. may be enumerated.

The recombinant vector of the invention was incorporated into E. coliDH5α [designation: pFPSm(S82F)/DH5α] and deposited at the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology (1-3, Higashi 1-Chome, Tsukuba City, IbarakiPref., Japan) on Dec. 12, 1997 as FERM BP-6551 under the BudapestTreaty.

(5) Production of the Geranyl Diphosphate Synthase

The geranyl diphosphate synthase of the invention can be obtained byculturing the transformant described above in a medium and recoveringthe synthase from the resultant culture.

The cultivation of the transformant of the invention in a medium iscarried out by conventional methods used for culturing a host.

As a medium to culture the transformant obtained from a microorganismhost such as E. coli or yeast, either a natural or synthetic medium maybe used as long as it contains carbon sources, nitrogen sources andinorganic salts assimilable by the microorganism and enables effectivecultivation of the transformant.

As carbon sources, carbohydrates such as glucose, fructose, sucrose,starch; organic acids such as acetic acid, propionic acid, citric acid;and alcohols such as glycerol, methanol, ethanol, propanol may be used.

As nitrogen sources, ammonia; ammonium salts of inorganic or organicacids such as ammonium chloride, ammonium sulfate, ammonium acetate,ammonium phosphate; other nitrogen-containing compounds; Peptone; meatextract; corn steep liquor, etc. may be used.

As inorganic substances, potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, magnesium phosphate, magnesium sulfate, magnesiumchloride, sodium chloride, iron(II) sulfate, manganese sulfate, coppersulfate, calcium carbonate, calcium chloride, etc. may be used.

When E. coli is used as a host, the cultivation is carried out usuallyunder aerobic conditions (such as shaking culture or aeration agitationculture) at 37° C. for 16 to 24 hrs. During the cultivation, the pH ismaintained at 6 to 8. The pH adjustment is carried out using aninorganic or organic salt, an alkali solution, a buffer or the like.During the cultivation, an antibiotic such as ampicillin or tetracyclinemay be added to the medium if necessary.

When a microorganism transformed with an expression vector having aninducible promoter is cultured, an inducer may be added to the medium ifnecessary. For example, when a microorganism transformed with anexpression vector having lac promoter is cultured,isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added tothe medium. When a microorganism transformed with an expression vectorhaving trp promoter is cultured, indoleacrylic acid (IAA) or the likemay be added.

As a medium to culture a transformant obtained from an animal cell as ahost, commonly used RPMI1640 medium or DMEM medium, or one of thesemedia supplemented with fetal bovine serum, etc. may be used.

Usually, the cultivation is carried out in the presence 5-10% CO₂ at 37°C. for 2 to 20 days. During the cultivation, an antibiotic such askanamycin or penicillin may be added to the medium if necessary.

As a medium to culture a transformant obtained from a plant cell as ahost, commonly used MS medium or this medium supplemented withkanamycin, various plant hormones, etc. is used. Usually, thecultivation is carried out at 20-30° C. for 3 to 14 days.

After the cultivation, the geranyl diphosphate synthase of the inventionis recovered by disrupting the microorganisms or cells if the synthaseis produced in the microorganisms or cells. If the geranyl diphosphatesynthase of the invention is produced outside of the microorganisms orcells, a culture supernatant is prepared by removing the microorganismsor cells by centrifugation or the like. Then, this culture (i.e. cellextract or culture supernatant) is subjected to conventional biochemicaltechniques used for isolating/purifying a protein. These techniquesinclude salting out, organic solvent precipitation, gel chromatography,affinity chromatography, hydrophobic interaction chromatography and ionexchange chromatography. These techniques may be used independently orin an appropriate combination to thereby isolate and purify the geranyldiphosphate synthase of the invention from the culture.

It should be noted that the geranyl diphosphate synthase of theinvention can have geranyl diphosphate synthase activity even when it isnot purified from the culture. Therefore, the cell extract or culturefluid may be used as a crude enzyme solution without purification aslong as it has the synthase activity.

(6) Preparation of Prenyl Diphosphate

According to the present invention, it is possible to accumulate GPP ina culture by culturing the host transformed with the DNA of theinvention and to prepare GPP by recovering the accumulated GPP.

According to the present invention, it is also possible to prepare GPPby allowing the enzyme of the invention to act on IPP or DMAPP which isa substrate for the synthase. In this method, the enzyme of theinvention is reacted with a reaction substrate in a solvent,particularly in an aqueous solution. Then, a prenyl diphosphate ofinterest is recovered from the reaction solution. As the enzyme, notonly a purified enzyme but also a crude enzyme which is semi-purified tovarious stages or an enzyme-containing material such as cultured cellsor a culture may also be used. Further, an immobilized enzyme which isobtained by immobilizing the above-mentioned enzyme, crude enzyme orenzyme-containing material by conventional methods may also be used.

As the substrate, IPP and/or DMAPP may be used. As the solvent for thereaction, water or an aqueous buffer such as Tris buffer or phosphatebuffer may be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the enzyme activity of mutant BstFPS andwild-type BstFPS.

FIG. 2 is a photograph of thin-layer chromatogram.

FIG. 3 is a graph showing the reaction product specificity of mutantBstFPS and wild-type BstFPS.

FIG. 4 shows comparison of amino acid sequences of farnesyl diphosphatesynthases.

BEST MODES FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described more specificallywith reference to Examples. However, the technical scope of theinvention is not limited to the following Examples.

Herein, amino acid residues are represented by the following one-letteror three-letter abbreviations.

A; Ala; alanine

C; Cys; cysteine

D; Asp; aspartic acid

E; Glu; glutamic acid

F; Phe; phenylalanine

G; Gly; glycine

H; His; histidine

I; Ile; isoleucine

K; Lys; lysine

L; Leu; leucine

M; Met; methionine

N; Asn; asparagine

P; Pro; proline

Q; Gln; glutamine

R; Arg; arginine

S; Ser; serine

T; Thr; threonine

V; Val; valine

W; Trp; tryptophan

Y; Tyr; tyrosine

Herein, the substitution of an amino acid residue is expressed usingone-letter abbreviations in the following order: “the amino acid residuebefore substitution”, “the position of the amino acid residue” and “theamino acid residue after substitution”.

For example, when Ser at position 82 is substituted with Phe, thissubstitution is expressed as “S82F”.

EXAMPLE 1 Preparation of a Plasmid Comprising FPP Synthase Gene

Bacillus stearothermophilus-derived FPP synthase (BstFPS) gene wassub-cloned into the NcoI-HindIII site of a plasmid vector pTV118N(commercially available from Takara Shuzo). This plasmid DNA isdesignated pFPS. The full-length nucleotide sequence of BstFPS gene isdisclosed by T. Koyama et al., (1993) J. Biochem., 113, 355-363 or ingenetic information databases such as DDBJ under Accession No. D13293.

EXAMPLE 2 Synthesis of an Oligonucleotide for Mutagenesis

The following oligonucleotide was synthetized in order to introduce amutation into the gene obtained in Example 1.

5′-CAT ACG TAC TTC TTG ATT CAT GAT GAT TTG-3′ (SEQ ID NO: 6)

The above oligonucleotide is designed so that (a) the Ser codoncorresponding to the amino acid residue at position 82 of BstFPS issubstituted with Phe codon; and (b) a restriction site for BspHI(5′TCATGA 3′) is newly introduced. The introduction of this BspHI sitedoes not cause any alteration due to degeneracy of codons in the aminoacid sequence encoded by BstFPS gene. Because of the introduction ofthis restriction site, it is possible to detect those plasmids intowhich a substitution mutation has been introduced by agarose gelelectrophoresis of plasmid DNA after BspHI digestion.

The synthesized oligonucleotide was phosphorylated in the followingreaction solution at 37° C. for 30 min and then inactivated at 70° C.for 10 min.

10 pmol/μl oligonucleotide 2 μl 10x kination buffer 1 μl 1000 mM Tris-Cl(pH 8.0) 100 mM MgCl₂ 70 mM DTT 10 mM ATP 1 μl H₂O 5 μl T4polynucleotide kinase 1 μl

EXAMPLE 3 Introduction of Substitution Mutation into the CodonCorresponding to the Amino Acid Residue at Position 82 of BstFPS Gene

Using the oligonucleotide synthesized in Example 2 as a primer, asubstitution mutation was introduced into the plasmid prepared inExample 1 according to the method of Kunkel. In the practice of thismethod, Mutan-K kit commercially available from Takara Shuzo was used.Experimental procedures were according to the protocol attached to thekit.

Briefly, a single-stranded DNA in which thymine in plasmid pFPS DNA hadbeen replaced with deoxyuracil was prepared using E. coli CJ-236 as ahost cell.

With this single-stranded DNA as a template, the primer DNA forcomplementary strand synthesis (i.e. the above oligonucleotide) wasannealed in the following solution.

Single-stranded DNA 0.6 pmol Annealing buffer 1 μl 200 mM Tris-Cl (pH8.0) 100 mM MgCl₂ 500 mM NaCl  10 mM DTT Primer DNA (from Example 2) 1μl H₂O to give a final volume of 10 μl

Subsequently, 25 μl of extension buffer, 60 units of E. coli DNA ligaseand 1 unit of T4 DNA polymerase were added to the solution and tosynthesize a complementary strand at 25° C. for 2 hrs. The extensionbuffer was composed of 50 mM Tris-Cl (pH 8.0), 60 mM ammonium acetate, 5mM MgCl₂, 5 mM DTT, 1 mM NAD and 0.5 mM dNTP.

Then, the reaction was terminated by adding thereto 3 μl of 0.2 M. EDTA(pH 8.0) and treating the resultant solution at 65° C. for 5 min.

EXAMPLE 4 Creation of a Transformant Whose Gene Has SubstitutionMutation in the Codon Corresponding to the Amino Acid Residue atPosition 82 of BstFPS Gene

E. coli DH5α was transformed with the DNA solution prepared in Example 3by the calcium chloride method as described below. Briefly, the DNAsolution was added to a suspension of DH5α competent cells treated with50 mM CaCl₂. Then, the suspension was put on ice for 30 min.

The resultant transformants were plated on an agar plate containingampicillin (a transformant selection marker), and cultured at 37° C.overnight. Plasmid DNA was prepared from a transformant havingampicillin resistance as a phenotype. After digestion with BspHI, theplasmid DNA was subjected to agarose gel electrophoresis to therebyselect substitution mutant pFPS plasmid which has a BspHI site withinthe BstFPS coding region from the resultant transformants.

Subsequently, the nucleotide sequence around the codon corresponding tothe amino acid residue at position 82 of BstFPS gene in the selectedsubstitution mutant pFPS plasmid was determined by the dideoxy method.As a result, a pFPS plasmid comprising a substitution mutant BstFPS gene(SEQ ID NO: 2) in which the Ser codon at position 82 (TCT) had beenreplaced with Phe codon (TTC) was obtained. This mutant is designatedS82F, and the plasmid pFPSm.

EXAMPLE 5 Determination of the Activity of Mutant BstFPS

Crude enzyme solutions were prepared as described below from twotransformants comprising the mutant BstFPS gene obtained in Example 4and wild-type BstFPS gene, respectively, and a transformant comprisingvector pTV118N alone.

Cells of each transformant cultured overnight in 2x LB medium wereharvested by centrifugation and suspended in a cell disruption buffer[50 mM Tris-Cl (pH 8.0), 10 mM β-mercaptoethanol, 1 mM EDTA). Thissuspension was sonicated and then centrifuged at 4° C. at 10,000 r.p.m.for 10 min. The resultant supernatant was thermally treated at 55° C.for 30 min to inactivate the prenyl diphosphate synthases derived fromE. coli. The thus treated supernatant was centrifuged under the sameconditions as described above to obtain a supernatant as a crude enzymeextract. This enzyme extract was reacted at 55° C. for 15 min in thefollowing reaction solution.

[1⁻¹⁴C]-IPP (1Ci/mol) 25 nmol Allylic diphosphate 25 nmol (DMAPP or GPPor FPP) Tris-Cl (pH 8.5) 50 mM MgCl₂ 5 mM NH₄Cl 50 mM β-mercaptoethanol50 mM Enzyme solution 50 μl H₂O to give a total volume of 1 ml

After the reaction, 3 ml of water-saturated butanol was added to thereaction solution to extract the reaction products into the butanollayer. To 1 ml of the resultant butanol layer, 3 ml of a liquidscintillator was added. Then, the mixture was subjected to thedetermination of radioactivity using a liquid scintillation counter.

The results are shown in FIG. 1. FIG. 1 is a graph showing the enzymeactivity of S82F mutant BstFPS and wild-type BstFPS. Sample Nos. 1, 4and 7 represent an enzyme prepared from a host comprising vector pTV118Nalone. Sample Nos. 2, 5 and 8 represents an enzyme prepared from a hostcomprising a gene encoding S82F mutant BstFPS. Sample Nos. 3, 6 and 9represent an enzyme prepared from a host comprising a gene encodingwild-type BstFPS. Further, sample Nos. 1, 2 and 3 represent the resultswhen DMAPP was used as an allylic substrate. Sample Nos. 4, 5 and 6represent the results when GPP was used as an allylic substrate. SampleNos. 7, 8 and 9 represent the results when FPP was used as an allylicsubstrate.

From FIG. 1, it is understood that the wild-type enzyme can use DMAPPand GPP as an allylic substrate but cannot use FPP. On the other hand,it is shown that the ability to use GPP as an allylic substrate isextremely lowered in S82F mutant enzyme.

Subsequently, a reaction solution was prepared separately in the samemanner as described above. Immediately after the reaction, 1 ml of apotato acid phosphatase solution [2 mg/ml potato acid phosphatase, 0.5 Msodium acetate (pH 4.7)] was added to the reaction solution, which wasdephosphorylated at 37° C. and then extracted with 3 ml of pentane. Theextract was analyzed by thin-layer chromatography [reversed phase TLCplate: LKC18 (Whatman); developer: acetone/water=9/1]. The developed,dephosphorylated reaction products were applied to Bioimage AnalyzerBAS2000 (Fuji Photo Film) to determine the positions and relativequantities of radioactivity.

The results are shown in FIGS. 2 and 3. FIG. 2 shows the TLC developmentpatterns of the dephosphorylated, mutant PstFPS reaction products whenindividual allylic substrates were used. For comparison, patternsobtained from samples prepared from hosts comprising wild-type BstFPSgene and a vector alone, respectively, are also shown. In this figure,“s.f.” represents the solvent front; “ori” represents the developmentorigin; “GOH” represents the position of geraniol standard sampledeveloped; and “FOH” represents the position of farnesol standard sampledeveloped. “Wild type” shows the results when wild-type BstFPS was used;“S82F” shows the results when mutant S82F BstFPS was used; and “vector”shows the results when an enzyme prepared from a host comprising avector alone was used. “n.d.” means that activity was not detected. FIG.3 is a graph showing the reaction product specificity of wild-typeBstFPS and mutant BstFPS. This graph shows GGPP, FPP and GPP generationratios when IPP and DMAPP were used as a substrate.

From the results shown in FIGS. 2 and 3, it is understood that whilewild-type BstFPS catalyzes a reaction to synthesize FPP specifically,S82F mutant BstFPS has been changed to catalyze a reaction to synthesizeGPP specifically. This means that S82F mutant BstFPS has been changed toan enzyme that can be called a geranyl diphosphate synthase.

Industrial Applicability

According to the present invention, a geranyl diphosphate synthase, agene encoding the synthase, a recombinant vector comprising the gene,and methods for preparing a geranyl diphosphate synthase and geranyldiphosphate, respectively, are provided.

The gene of the invention is useful since it is applicable to metabolicengineering and enzyme engineering aiming at the synthesis ofmonoterpenes.

Free Text to the Sequence Listing

SEQ ID NO: 1: Xaa represents Val, Leu, Ile, Thr, Asp, Glu, Asn, Gln,Lys, Arg, Cys, Met, Phe, Tyr, Trp, His or Pro.

SEQ ID NO: 6: Oligonucleotide which is designed based on the amino acidsequence of FPP synthase and has a BspHI site.

What is claimed is:
 1. An isolated recombinant protein selected from thegroup consisting of: a) a protein comprising the amino acid sequenceshown in SEQ ID NO: 1, and which has geranyl diphosphate synthaseactivity; and b) a protein which comprises the amino acid sequence shownin SEQ ID NO: 1 having deletion, substitution or addition of one aminoacid outside of conserved regions I, II, III, IV, and V of said protein,and which has geranyl diphosphate synthase activity.
 2. An isolated genecoding for the recombinant protein selected from the group consistingof: a) a protein comprising the amino acid sequence shown in SEQ ID NO:1, and which has geranyl diphosphate synthase activity; and b) a proteinwhich comprises the amino acid sequence shown in SEQ ID NO: 1 havingdeletion, substitution or addition of one amino acid outside ofconserved regions I, II, III, IV, and V of said protein, and which hasgeranyl diphosphate synthase activity.
 3. An isolated geranyldiphosphate synthase gene comprising the nucleotide sequence shown inSEQ ID NO:
 2. 4. A recombinant vector comprising the gene of claim 2 or3.
 5. A transformant transformed with the recombinant vector of claim 4.6. A method of preparing a geranyl diphosphate synthase comprisingculturing the transformant of claim 5 in a medium and recovering thegeranyl diphosphate synthase from the resultant culture.
 7. A method ofpreparing geranyl diphosphate comprising culturing the transformant ofclaim 5 in a medium and recovering geranyl diphosphate from theresultant culture.
 8. A method of preparing geranyl diphosphatecomprising allowing a culture of the transformant of claim 5 to act onisopentenyl diphosphate or an isomer thereof.
 9. An isolated recombinantprotein comprising the amino acid sequence shown in SEQ ID NO: 1, andwhich has geranyl diphosphate synthase activity.
 10. An isolatedrecombinant protein comprising the amino acid sequence shown in SEQ IDNO: 1 having deletion, substitution or addition of one amino acidoutside of conserved regions I, II, III, IV, and V of said protein, andwhich has geranyl diphosphate synthase activity.
 11. An isolated genecoding for the recombinant protein comprising the amino acid sequenceshown in SEQ ID NO: 1, and which has geranyl diphosphate synthaseactivity.
 12. An isolated gene coding for the recombinant proteincomprising the amino acid sequence shown in SEQ ID NO: 1 havingdeletion, substitution or addition of one amino acid outside ofconserved regions I, II, III, IV, and V of said protein, and which hasgeranyl diphosphate synthase activity.